Diamino and substituted diamino tetrahydroxy cyclohexanes



Patented June 19, 1951 UNITED STATES- PATE DIAMINO AND TETRAHYDROXY CYCLOHEXANES" Robert L. Peck, Plainfield, N. J., assignor to Merck & 00., Inc., Rahway, N. J., a corporation of New Jersey No Drawing.

This application is'a continuation-impart of my pending application Serial No. 624,333, filed October 24, 1945, now abandoned.-

This invention relates to cyclohexane compounds, and particularly totetrahydroxy cyclohexanes having .two amino, carbamido, or guanido substituents in the cyclohexane ring, and to acid salts and acylderivatives of such cyclohexanes. These substances have antibacterial propstrucural formula 5 no c \CH0H 110- H 11-)! wherein the substituents X are-the same and of To simplify reference to these primary substances and their acid salts and acyl derivatives streptidine is a strongly basic substance forming crystalline acid, salts and acyl derivatives. It can be prepared from substantially pure streptomycin hydrochloride, such as streptomycin hydrochloride purified by selective adsorption and Application February26, 1946, Serial No. 650,392

" Claims. (01. 260553) elution as fully disclosed in applicants pending application Serial No'. 601,337, filed 'June 23, 1945,

or obtained by regeneration of the helianthine' salt of streptomycin described in a publication by Kuehl, Peck, Walti, and Folkers in Science 102,

34-35 (July 13, 1945).

It has been discovered in accordance with the present invention that when st-reptomycin hydrochloride is treated with a mineral'acid such as hydrochloric acid, sulfuric acid, and the like 'in aqueous lower aliphatic alcohol, or mixed aqueous-lower aliphatic alcohol solution, there is a cleavage of the streptomycin molecule result: ing in the formation of the corresponding mineral acid salt of streptidine. As streptidine is relatively thermostable the reaction can be'carried out at elevated temperature, for example, by heating the reaction mixture at about 120? C., under pressure, for about two hours; however, it will also be understood that the reaction can be carried out at room temperature by allowing the reaction mixture to stand for about 18-20 hours.

v sis.

streptidine is recovered from the reaction mixture in the form of a readily crystallizable salt such as the picrate salt in the manner fully set forth in the examples; and from the crystalline product thus obtained other organic and inorganic acid salts as well as acyl derivatives are obtained as described in the examples. It will be noted, however, that when streptomycin hydrochloride is reacted with concentrated sulfuric acid in absolute methanol, the sulfate of streptidine separates directly in crystalline form.

When streptidine in the form of an acid salt such as its hydrochloride is hydrolyzed by heating for about one hour in dilute aqueous alkali, preferably in an oxygen free (nitrogen) atmosphere, it is converted to strepturea, which .is recovered as a crystalline product.

Prolonged alkaline hydrolysis of either streptidine or strepturea in an oxygen free (nitrogen) atmosphere yields streptamine. As the reaction is brought about by the OH- ion in solution, it is preferable, to use the alkali in dilute solution although solutions containing up to about 30% sodium hydroxide have been used successfully. It will be understood in this-connection that any alkali metal or alkaline earth metal hydroxide in aqueous solution can be used for this hydroly- Streptamine is formed in the hydrolysis solution as the free base, and upon neutralization of the excessalkali it can be recovered as such, or preferably as a streptamine acid salt such as the crystalline streptamine sulfate. Streptamine sulfate can in turn be converted to NT OFFICE v of ether.

3 other inorganic or organic acid salts, acyl derivatives, or free base by procedures fully set forth in the examples.

The following examples illustrate methods of carrying out the present invention, but it is to be understood that these examples are given by way of illustration and not of limitation.

EXAMPLE I streptidine picrate About 320 mg. of streptomycin hydrochloride regenerated from pure streptomycin helianthate is dissolved in about 2 cc. of water and 1.0 cc. of concentrated hydrochloric acid. The solution is heated in a sealed ampoule at 120 for 2 hours. The hydrolysis mixture is then evaporated to dryness, dissolved in methanol, filtered through activated charcoal, and again evaporated to dryness. The residue, about 178 mg., is separated into an ethanol-soluble fraction, about 89 mg. The ethanol-soluble fraction is dissolved in about cc. of water, about 118 mg. of picric acid (containing 10% moisture) is added, and solution is effected by heating. On cooling, long needle crystals separate. After recrystallization according to conventional methods, the crystals melt at about 283-284 (micro-block) with decomposition. Polarographic analysis shows the presence of 61.1% plcric acid. The elementary analyses of a sample dried at in vacuo agree with the formula CaH1aNsO4.2CsHaN3O7.2I-I:O: Calcd.: C, 31.75; H, 3.73; N, 22.22. Found C, 31.65, 31.96; H, 3.47, 3.61; N, 22.01. Analyses on a sample dried at 56 in vacuo agree with the formula of the anhydrous streptidine picrate, CaH1sNsO4.2 CsH3N3Ov. Calcd.: C, 33.34; H, 3.36; N, 23.33. Found: C, 33.21; H, 3.03; N, 22.75. a

A sample of streptidine picrate crystallized from water as the dihydrate was recrystallized EXAMPLE II streptidine hydrochloride About 234 mg. of streptidine picrate was suspended in 3 cc. of methanol and 0.1 cc. of concentrated hydrochloric acid was added causing solution to occur. Addition of about 25 cc. of acetone caused the separation of a white precipitate which was washed with acetone and dried, iving 112mg. of dry powder. pears to be a dihydrochloride. It melts at about 170-210 (micro-block) with decomposition. The material is hygroscopic and is dried'at 100 in vacuo for analysis.

Anal. Calcd. for CsH1sNsO4.2HC1Z C,28.66; H, 6.01; N, 25.07. Found: C, 29.31; H, 6.57; N, 25.16.

The dihydrochloride, about 26 mg., on treatmentgwith excess diazomethane in methanol solution yieldsa-1:rystalline-.prfluct n addition The yield is about 12 mgi'of 'sinall needles. The crystals melt to a viscous syrup at about 158-160 (micro-block), and give off gas up to 200. This appears to be streptidine monohydrochloride.

Anal. Calcd. for CsHiaN604.HCl: C, 32.16; H, 6.41; N, 28.13. Found: C, 31.88; H, 6.69; N, 25.20.

EXAMPLE-III streptidine Itelianthate A hot solution of about 1 g. of methyl orange This apin 70 cc. of water is added to a solution of about 421 mg. of streptidine hydrochloride in 5 cc. of methanol. A voluminous, finely crystalline, orange-brown precipitate separates. This, after collection by centrifugation, is recrystallized from aqueous methanol. A yield of about 1.1 grams of streptidine hellanthate is obtained. The crystals sinter at about 225, and melt at about 246 250 (micro-block) with decomposition.

A sample drled'at 56 in vacuo gave the following analytical results: Calcd. for

. CaaHnNnOroSsrZHzO C, 47.57, H, 5.77. Found: C, 47.25; H, 5.60. A sample dried at in vacuo gave the following results: Calcd. for CuHuNuOmSz: C, 49.53; H, 5.54. Found: C, 49.31; H, 5.30.

EXAMPLE IV v streptidine d-oamphorsulfonate About 1.048 g. of streptidine hydrochloride is dissolved in 3 cc. of methanol. Slightly more than the equivalent amount of sodium d-camphorsulfonate, about 1.6 g., dissolved in 3 cc. of methanol is added. On standing, sodium chloride separates and is filtered 01!. The filtrate is diluted with about 10 cc. of absolute ethanol, whereupon a voluminous precipitate of white needles separates. The yield in the first crop'is about 1.49 g. The recrystallization of streptidine d-camphorsulfonate is carried out in 50% methanol-ethanol, and gives eedles melting at about -188 (micro-block).

Analysis of a sample dried at 56 in vacuo gave the following values: Calcd. for

CsHisNsO4.2CioI-I1504S H2O C, 45.15; H, 7.04; N, 11.28. Found: C, 45.42; H, 7.10; N, 11.41. Analyses of the same sample dried at 100 in vacuo gave the following values: Calcd.: for C8Hl8N604'2Cl0Hl604SZ C, 46.27; H, 6.93. Found: C, 46.25; H, 6.52.

EXAMPLE v v streptidine chloroplatinate About 21 mg. of streptidine hydrochloride is dissolved in 0.5 cc. of water and mixed with 1.0 cc. of a 10% aqueous solution of chloroplatinic acid.

The clear solution is diluted to about 12 cc. with absolute ethanol and allowed to stand for some time in the cold room. Orange-yellow crystals are deposited which after drying weigh about 32 mg. These crystals begin to decompose at about 231 (micro-block). The crystals formed in the above manner appear to be pure streptidine chloroplatinate as is shown by elementary analyses obtained on such preparations.

Anal. Calcd. for CsHisNsOaHzPbCleZ C, 14.29; H, 3.00; N, 12.50; Pt,29.04. Found: C, 14.38; H, 3.08; N, 11.97; Pt, 28.82.

EXAMPLE VI Octaacetul streptidine About 1.01 g. of streptidine hydrochloride, about 494 mg. of fused sodium acetate, and 20 cc. of acetic anhydride are refluxed together for one hour. The acetylation mixture is concentrated to dryness in vacuo. To the solid residue water is added and the insoluble crystalline acetyl derivative is filtered off and dried giving about 152 g. of needle crystals. The crystalline product is recrystallized from chloroform-petroleum ether mixture giving about 1.22 g. of needle crystals melting at about 260 (micro-block).

' below 300.

Analyses of typical preparations are as follows:

Calcd.'for Cad-134M012: C, 48.15; H, 5.73; N, 14.04;

acetyl, 57.52. Found: (1) C, 48.39; H, 5.09; N, 13.72. (2) C, 48.20; H, 5.73; N, 13.75; acetyl, 59.2. (3) .C, 48.40; H, 5.73; N, 14.84; acetyl, 57.0.

Ultraviolet absorption spectrum determination showed a maximum at about 2200 A., l% =about' 400, a maximum at about 2525 A, 1%=about 630, and a minimum at about 2335 A, 1%= about 295.

I EXAMPLE vn Streptz'dine dihydroiodide About 304 mg. of octaacetyl 'streptidine, 150 mg.

. hydrolysis. The molar ratio of aminoniaevolved to carbon dioxide evolvedin thisfhydrolysis is 2:1. carbon dioxide are expected to be produced in the complete hydrolysis for each mole of streptidine. The product left after. the loss of ammonia and .carbon dioxide from streptidine is called streptamine. Milder hydrolysis, for the periodof one hour, yields only two molesofammonia and no carbon dioxide. The product obtained under theseconditions is the urea-type; compound strepturea. Further hydrolysis ofthe latter of red phosphorus, 576 mg. of iodine, and 5 cc. of

hydriodic acid, sp. 'g. 1.7,are mixed in atbflmb tube and heated at 160-190 for about '7' hours.

The tube is opened and the contents diluted with water and filtered.- The filtrate is evaporated to dryness leaving a semi-crystalline-residue. This is dissolved in methanol; addition of ether causes the separation of white crystals of streptidine dihydroiodide. A sample was analyzed, giving the following results.

Anal; Calcd.' for- C8H18N604.2HII C, 18.54;

H, 3.89; N, 16.22; I, 48.99. Found: C, 19.43

H, 3.96; N, 15.75;I, 48.52. 1

EXAMPLE VIII Strepttdine sulfate About 1 g. of streptidine picrate was dissolved in 50 cc. of methanol and excess concentrated sulfuric acid, 1.0 cc., was added. White needle crystals separated on standing for ashort time.

These crystals appear to contain one mole of methanol of crystallization.

Anal.

The crystals are recrystallized from'water containing excess sulfuric, acid .byaddition ofacetone, and give the-alcohol-jfree streptidine sulfate.

The yield of three-timesrecrystallized'product is about 300 mg. A sample was 'driedat 100 in. vacuo for analysis. j

Anal. Calcd. for CaH1aNsO4l-I2SO'4I C, 26.66; H, 5.60;'. N, 23.33. Found: C, 26.91; H, 5.66;

Streptidinesulfate exhibits no melting point EXAMPLE I): Stre'ptcmine sulfate (hydrolysis of streptidine) sis solution was filtered away from the barium carbonate formed, and the barium carbonate was dissolved in dilute hydrochloric acid and converted to barium sulfate yielding 1.144 g. This corresponds to the formation of 1.82 moles of carbon dioxide per mole of streptidine during the Calcd. for C8H18N6OLH2SOLCH3OHZ' o, 27.55;,H. 6.17: N, 21.42. Found: o, 27.78;

This is, within experimental error, approximately 4 moles per mole, which is the expected value. The hydrolyyields, of course, strep-tamine, carbon dioxide, and ammonia.

The filtrate above obtained after removal o bariumcarbonate was then treated'with that amount'of' standard sulfuric acid calculated to neutralizethe barium hydroxide leftin the'filtrate. The barium sulfate was filtered off and the filtrate was frozen'and dried from the frozen .state under vacuum, yielding 710mg. of buff amorphous solid after further drying at 56 for' about an hour. About 277 mg. of the residue thus obtained was ground up in about 5 'cc. of water,

whereuponf'needle crystals were formed. "These were recrystallized fromabout'40cc. of boilin water. After removal of a first crop-of crystals, 158 mg, further crops could be'obtainedfby addition of methanol to the-mother liquors. The crystals did not: show any melting point up to 300.

They represent the normal-sulfate of streptamine, since analyses agree with this, and

since the sulfate streptamina I I Calo. for (26H14N2O4.H2SC)'4: C; 26.12; H,

can be converted to hexaacetyl Anal.

5.84; N, 10.14; Found: C,-26.36; H,'6.09; N, 9.91.

EXAMPLE x Streptzdz'ne (free base) 7 About 246 mg. of streptidine sulfatedissolved' in 4000. ofhot water is treated with3,28 cc. of

0.372*N barium hydroxide. The barium sulfate.

' .the crystalline free base streptidine. This prodnot, when heated on a micro-block sinters slightly formed is removed by filtration and the aqueous filtrate. is concentrated to dryness-from the frozen state. Treatment of the residue with methanol ,yields a'crystalline product and recrystallization from aqueous methanol yields about mg. of

at about 205 C., begins to decompose at about 225? C. and appears to decompose completely between 280-290 C. A

* EXAMPLE XI Streptamz'ne hydrochloride A quality of streptamine sulfate is treated with a stoichiometric equivalent of barium chloride.

A metathetical reaction takes place with the formation of a precipitate of barium sulfate which is filtered off. The filtrate is concentrated to dryness and a small amount of methanol is added. The addition of methanol causes forma tion of needle crystals of streptamine hydrochloride which after removal of the methanol and drying have a melting point of about 210-'-'220 C. with decomposition.

' EXAMPLE XII Streptamine (free base) I I About 1.24 g. of streptamine sulfate,

CsI-I14N2O4.H2SO4 was suspended in 50 cc. of water and heated to boiling. A. slow stream of nitrogen was kept Four moles-of ammonia and two molesof.

passing over the surface of the hot suspension. To the boiling suspension was added 19.36 cc. of 0.465 N barium hydroxide. The suspension was kept boiling for about ten minutes in order to complete the metathetical reaction. After coolwere collected. A further amount could be obtained from the mother liquors of the first .two crops by conventional procedures. The crystals sintered slightly at 205, began to decompose at about 210 (slight darkening) and were completely black at about 250 when heated on the micro-block. The crystals did not melt up to 290,

however.

Anal. Calcd. for CsH14NzO4. /2HzO: C, 38.49; H, 8.08; N, 14.97. Found: C, 38.65; H, 7.62; N, 15.43.

EXAMPLE XIII strepturea About 371 mg. of streptidine hydrochloride were treated with 10 cc. of 0.3721 N barium hydroxide and refluxed for one hour in a stream of nitrogen. Then 4.83 cc. of 0.7711 N sulfuric acid was added to neutralize exactly the barium hydroxide, and the barium sulfate formed was removed by filtration. The filtrate was evaporated to dryness and extracted with methanol. The insoluble material, 210 mg., was then dissolved in about 5 cc. of hot water and allowed to cool slowly after adding about 8 cc. of methanol. Clusters of needles of strepturea separated on cooling. The crystals darkened at 270 and decomposed at about'290-300. Strepturea appears to be a neutral substance.

Anal. Calcd. for CaHmN-iOe: C, 36.36; H, 6.10; N, 21.20. Found: C, 35.85; H, 6.43; N, 20.60.

EXAIVIPLE XIV streptamine-p-hydrozyphenyZ-azobeizgenep'-sulfonate About 72 mg. of free streptamine was dissolved in 0.5 cc. of water and treated with 300 mg. of phydroxyphenyl azobenzene p sulfonic acid, whereupon a voluminous precipitate of needles separated. These were recrystallized from hot water. The crystals decompose at about 270-300 micro-block). They were dried at 100 in vacuo for analysis.

Anal. Calcd. for C3oH34N6O12S2: C, 49.04; H, 4.67; N, 11.44. Found: C, 48.82; H, 4.67; N, 11.61.

EXAMPLE XV streptamine picrate Calcd. for CaH14N2O4.2CaHaNsO7I C,

T 8 33.97; H, 3.17; N, 17.61. 3.42; N, 17.52.

Found: C, 33.57; H,

EXAMPLE XVI streptamine hydroiodide About 139 mg. of streptamine sulfate was heated in a bomb tube with 150 mg. of red phosphorus and 5 cc. of hydriodic acid" (sp. g. 1.7) for 15 hours. The contents of the tube were diluted. filtered and the filtrate was concentrated to dry mess. The residue was dissolved in methanol. Crystals of streptamine hydroiodide were deposited on addition of ether to the methanol solution. The crystals melted with decomposition at about 250-260 (micro-block).

Anal. Calcd. for CaH14N-2O42HI: C. 16.60; H, 3.72; N, 6.46; I, 58.48. Found: C, 15.09; H, 3.55; N, 6.68; I, 57.17.

EXAMPLE XVII H exaacetyl streptamine About 81 mg. of streptamine sulfate, 50 mg. of fused sodium acetate, and 5 cc. of acetic anhydride were heated under reflux for one hour. At the end of this time the solution was evaporated to dryness in vacuo. The residue was extracted with chloroform yielding about 40 mg. of white needles soluble in chloroform. These crystals show a transition point at about 250-251 (microblock) which appears to be a change of crystal form to long fine needles. At this temperature there is a semi-solid mixture of needles and semi-solid material which all appears to go over into the needle form by 285-295. No true melting is observed up to 300. This sample was recrystallized from chloroform solution by addi tion of ether, giving about 27 mg. of needles which showed the same melting point behavior. These crystals appear to be one isomer of hexaacetyl streptamine characterized by chloroform solubility.

Anal. Calcd. for C6HaNzO4.6CHaCO: C, 50.20; H, 6.09; N, 6.50; acetyl, 60.0. Found: C, 51.00; H, 6.05; N, 6.03; acetyl, 61.1.

From the chloroform insoluble residue left after extraction of the acetylation mixture with chloroform, there was separated about 90 mg. of long needles by suspending the residue in chloroform and mechanically separating the needles (dispersed in chloroform) from the sodium chloride (heavy crystals which rapidly settled to the bottom out of suspension). These crystals also showed a change in crystal form when heated, changing to long fine needle's at about 240-247 (micro-block) These crystals did not melt up to 300 (micro-block). They were recrystallized from ethanol, giving 43 mg. of crystals with about the same melting point behavior. They appear to be an isomer of hexaacetyl streptamine characterized by chloroform insolubility.

Anal. Calcd. for CsHaN204.6CH3COZ C, 50.20; H, 6.09; N, 6.50; acetyl, 60.0. Found: C, 50.08; H, 6.10; N, 6.17; acetyl, 56.9.

EXAMPLE XVIII N,N-diacetyl streptamine Hexaacetyl streptamine, about 38 mg., is dissolved in 10 cc. of absolute methanol, cooled to about 0, and gaseous ammonia is passed into the solution until saturation occurs.

. tion in a stoppered flask is then allowed to warm to room temperature and stand for three hours. The solvent is removed at reduced pressure at a low temperature. The crystalline residue left The solu L position.

A sample dried at 56 in vacuo gave the following analytical results: Calcd. for

CsH12N2O4.2CHaCO.H2O

c, 42.85; H, 7.19; N, 10.00. Found: 0, 42.91; H, 7.07;'N,10.07 v I EXAMPLE XIX Hzabenzoyl streptamine About 744 mg. of streptamine hydrochloride was refluxed for 10 minuteswith 6 ml. of dry pyridine and 3.5 ml. of benzoyl chloride. After cooling, the mixture was diluted with water and extracted with chloroform. The chloroform extract was washed with dilute hydrochloric acid, dilute sodium bicarbonate, and water. The

- chloroform extract after drying was evaporated to about a volume of about 15 ml. and mixed with three volumes of ether. Separation of a crystalline precipitate of hexabenzoyl streptamine was rapid. The crystals weighed 780 mg. and did not melt up to 300.

Anal. Calcd. for CeHaN2O4(CeH5CO)6Z C. 71.81; H, 4.77; N, 3.49; benzoyl, 78.5. Found: C, 72.06; H, 5.03; N, 3.35; benzoyl, 70.5.

EXAMPLE XX N,N'-dibeneoy'l streptamine About 250 mg. of hexabenzoyl streptamine was heated with 60 ml. of absolute methanol containing a trace of sodium methoxide and 5 ml. of pyridine to increasesolubility of the hexa-' benzoate. After a short period of heating, the solid hexabenzoate was all dissolved and the solution was evaporated to dryness. The residue was dissolved in warm methanol, filtered, and diluted with water. On standing, long needle crystals separated. After two crystallizations the crystals of-N,N'-dibenzoyl streptamine weighed 160 mg., M. P. 293-295 (micro-block). could be obtained from the mother liquors. The sample was dried at 100 in vacuo for analysis.

Anal. Calcd. for CsH12N204 (CeHsCO) 2 I C. 62.16; H, 5.74; N, 7.25. Found: C, 61.79; H, 5.88; N, 7.21.

Modifications may be made in carrying out Further amountsthe present invention without departing from the spirit and scope thereof and the invention is to be limited only by the appended claims.

I claim: 1. A substance of the class consisting of diamino, dicarbamido, and diguanido tetrahydroxy cyclohexanes and acid salts and acetyl and benzoyl derivatives thereof.

2. The diamino tetrahydroxy cyclohexane, streptamine.

3. The dicarbamido tetrahydroxy cyclohexane,

- strepturea. v

4. The diguanido tetrahydroxy cyclohexane, streptidine.

1 5. The process mycin hydrochloride with a mineral acid in a solvent of the class consisting of water. lower aliphatic alcohols, and aqueous-lower aliphatic alcohol mixtures, thereby forming the diguanido tetrahydroxy cyclohexane, streptidine.

6. The process that comprises treating streptomycin hydrochloride with a mineral acidin a solvent of the class consisting of water, lower aliphatic alcohols, and aqueous-lower aliphatic alcohol mixtures, thereby forming the diguanido tetrahydroxy cyclohexane, streptidine, and recovering streptidine from the reaction-mixture in the form of an acid salt. v

7. The process that comprises treating streptomycin hydrochloride with a mineral acid at about 120 C., under pressure, for about two hours, in a solvent of the class consisting of water,

lower aliphatic alcohols, and aqueous-lower aliphatic alcohol mixtures, thereby forming the diguanido tetrahydroxy cyclohexane, streptidine.

9. The diguanido tetrahydroxy 'cyclohexane dihydrochloride, streptidine hydrochloride.

10. The diamino tetrahydroxy cyclohexane dihydrochloride, streptamine hydrochloride.

ROBERT L. PECK.

' 'REFERENCES CITED The following 'referencesare of record in the file of this patent:

, Jan. 1944, pp. 66-69.

I 1945, page 339.

Carter et al.: J. Biol. Chem., vol. .160, Sept.

that comprises treating strepto- 

1. A SUBSTANCE OF THE CLASS CONSISTING OF DIAMINO, DICARBAMIDO, AND DIGUANIDO TETRAHYDROXY CYCLOHEXANES AND ACIDS SALTS AND ACETYL AND BENZOYL DERIVATIVES THEREOF. 